Acutely administered morphine produces antinociception via mu opioid receptor (MOR)-mediated intracellular changes that include a decrease in calcium concentration, and a decrease in the activity of adenylyl cyclase and protein kinases. Chronic administration of morphine results in tolerance to its antinociceptive effects and a reversal in the direction of these intracellular events. Our recent studies have implicated protein kinases, especially cAMP-dependent protein kinase (PKA) and protein kinase C (PKC), as well as other steps in the phosphatidylinositol cascade in morphine tolerance. We have reported that inhibition of either PKA or PKC, blockade of phospholipase C or blockade of 1P3 receptors all cause a reversal of morphine tolerance. In other studies we have elucidated the involvement of ATP-gated potassium channels in the actions of acute and chronic morphine. Again we found diametrically opposite effects in the acute versus chronic treatment regimens. The overall goal of the proposed studies is to utilize pharmacological, biochemical and anatomical approaches to determine the critical cellular events underlying the tolerance that develops to morphine-induced antinociception. We have proposed a comprehensive model that includes the steps in the MOR-mediated signal transduction cascade that we hypothesize are involved in tolerance to morphine-induced antinociception. The proposed experiments will test our hypotheses that constant phosphorylation of proteins is required for the maintenance of morphine tolerance and that inhibition of protein-kinase-mediated phosphorylation or upstream steps in their signaling cascades reverses morphine tolerance. We propose to elucidate the mechanisms that lead to the reversal of analgesic tolerance by examining mu opioid receptor levels and phosphorylation state, G-protein activation, adenylyl cyclase activity, and the phosphorylation state of L- and N-type calcium channels.